The mechanism of action of carcinostatic agents, such as BCNU and CCNU, will be investigated. These nitrosourea derivatives have been shown to inhibit DNA synthesis in vivo. We have shown that in vitro these agents, and the isocyanates derived from their decomposition, 2-chloro-ethyl isocyanate and cyclohexyl isocyanate, inhibit one of the two forms of purified non-mitochondrial DNA polymerase (I and II) from rat liver and hepatomas and HeLa (S3) cells. DNA polymerase I, an enzyme that does not change with proliferation, is insensitive to these agents. However, DNA polymerase II is inhibited by these agents and its activity correlates positively with the rate of proliferation. In this study we will determine in vitro the mechanism of inhibition of purified DNA polymerase II by these agents. This will include studies of: The essential substituents of the agents for inhibition, the kinetics and stoichiometry of the reaction(s) and the site(s) and mechanism(s) of binding of the agents to homogeneous DNA polymerase II. We will also investigate the effect of these agents on other key enzymes for DNA biosynthesis, such as ribonucleotide reductase, thymidylate synthetase etc. We will investigate the effect of these agents in vivo on DNA polymerases in regenerating rat liver, and also in vitro in HeLa cells in culture. BIBLIOGRAPHIC REFERENCES: B. Baril, E. Baril, J. Laszlo and G. Wheeler. Inhibition of Rat Liver DNA Polymerases by Nitrosoureas and Isocyanates. Cancer Res. 35; 1-5 (1975). R. Chiu and E. Baril. Nuclear DNA Polymerases and the HeLa Cell Cycle. J. Biol. Chem. November l975, in press.